The present invention relates to a pH sensor for the visual or optical indication of the pH of a sample, the sensor having a mechanically stable support and an indicator dye immobilized thereby. The invention further relates to a process for fabricating a pH sensor for visual or optical indication of the pH of a sample, in which a pH indicator dye is immobilized by a mechanically stable support, such that the indicator dye does not appreciatively bleed when contacted with the sample. In addition, the invention also relates to the use of a pH sensor of this type. Finally, the invention also relates to an efficient method of selecting pH indicator dyes and/or dyes ratio for obtaining a pH indicator dye mixture which is sensitive to a subtle change in pH.
The optical measurement of the pH goes back to the finding that certain dyes, (such as, for example, litmus and tea) react to the pH of a sample by changing their color and are thus able to indicate the pH of a sample. A selection of suitable dyes (indicators), together with the pH ranges within which they change color, is found, for example, in xe2x80x9cIndicatorsxe2x80x9d, E. Bishop, Pergamon Press, 1972, chapter 3, which is incorporated herein by reference.
The first pH-sensitive strip materials were obtained by immersing a paper strip in a dye solution. The dye in color strips of this type is bound to the support by absorption. This has the drawback that the dye can be washed out by the sample, which makes such strips unsuitable for applications wherein indicator contamination is unacceptable and for continuous processes. In addition, in many cases, upon drying such strips loose their pH indicative color.
By means of chemical or physical immobilization of dyes washing out can be prevented. Immobilized pH-sensitive materials are therefore suitable for the continuous measurement of pH and for applications wherein indicator contamination are unacceptable.
An early study of bacterial vaginosis (BV) involved comparisons of the pH of vaginal fluids of women known to be suffering from BV with those known to be free of the disease. Gardner, H. L., et al., Am. J. Obstet. Gynecol. 69:962 (1955). All of the BV positive women in the study were determined to have a vaginal fluid pH greater than 4.5, and 91% of these women had a vaginal fluid pH greater than 5.0. Of the normal (disease-free) women in the test, 92% were found to have vaginal pH between 4.0 and 4.7. The conclusion drawn from the study was that a vaginal pH equal to or greater than 5.0 in conjunction with other clinical criteria was indicative of the presence of BV.
Subsequent studies culminating by Amsel, R., et al., Am. J. Med. 74:14-22 (1983), resulted in a reduction of the pH threshold for BV to 4.5, and established the remaining criteria as vaginal fluid homogeneity, the whiff test (treatment with alkali followed by an olfactory test to detect for an amine odor), and the presence of clue cells. These are commonly referred to as the Amsel clinical criteria for BV. The conclusion was based on a study group of 397 women in which 81% of BV positive women were found to have a pH greater than 4.5 while only 23% of the normal women were found to have a vaginal fluid pH greater than 4.5.
Studies subsequent to the report by Amsel et al. have now adjusted the pH threshold to 4.7. One of these is the study of Holst, E., J. Clin. Microbiol. 28:2035-2039 (1990), in which 100% of the women diagnosed as BV positive by the Amsel criteria were reported to have vaginal fluid pH greater than 4.7. Another is the study by Eschenbach, D. A., Am. J. Obstet. Gynecol. 158(4):819-828 (1988), in which all 257 women in the study group who had at least 20% clue cells were shown to have a vaginal fluid pH greater than or equal to 4.7, leading to the conclusion that a threshold value of 4.7 correlated best with the other clinical evidence of BV. Krohn, M. A., et al., J. Clin. Microbiol. 27(6):1266-1271 (1989), also verified the correlation between the vaginal fluid pH threshold of 4.7 and the presence of clue cells, and Holmes, K. K., and coworkers further confirmed the pH 4.7 threshold as an indicator of BVxe2x80x94Holmes, K. K., et al., eds., Sexually Transmitted Diseases, McGraw-Hill, New York (1990), Chapter 46:527-545 (Holmes, K. K., et al.), and Chapter 47:547-559 (Hillier, S. L., et al.).
Colorimetric tests for elevated vaginal fluid pH have used nitrazine yellow as an indicator. Nitrazine yellow is a monoazo dye that is bright yellow at pH 6.0 and bright blue at pH 7.2, and has a grey-green midpoint at pH 6.6. In the range of interest for testing vaginal fluid, however, which is approximately 3.5 to 6.0, the change occurs in subtle progressions of grey-green that are difficult to interpret.
pH thresholds are useful indicators in diagnosing a variety of other biological conditions, in both humans and animals, and a large number of colorimetric indicators are known and commercially available. Selection of the appropriate indicator is not always a simple task, however, and the choices are often limited, particularly when a specific color change is desired and when stability of the indicator is a consideration.
Normal bovine milk, for example, has a pH of 6.5 to 6.8, and it has been reported (N. Z. J. Sci. Technol. 27:258 (1945)) that bovine milk with a pH greater than 6.8 may indicate the presence of bovine mastitis. The difficulties of detecting a pH deviation of such a small magnitude using conventional paper indicator strips are discussed above.
A study specifically directed to bovine mastitis was reported in J. Dairy Sci. 68: 1263-1269 (1985). The purpose of the study was to determine the suitability of using absorbent blotting paper impregnated with the pH indicator bromthymol blue to test the pH of bovine milk as a method of detecting subclinical bovine mastitis. Milk was added to the indicator-treated paper, and the color of the pH indicator spot was scored on a scale of 1 to 4, where 1 (pale green) was assessed as normal (negative), and 2, 3 and 4 (increasing from moderate green to dark blue-green) were considered abnormal (positive). The pH of the milk was also determined electronically with a carefully calibrated pH meter. The test results illustrated the difficulty in defining accurately the color of the test area: the predictive value of a positive colorimetric test ranged from 49% to 52% (i.e., 51% to 48% of the results were false positives).
As expected, an increase in the test score was accompanied by an increase in the severity of mastitis as defined by other diagnostic measures. However, in milk from animals with less severe mastitis, the considerable overlapping of results highlighted the possible error in interpretation of indicator scores. The wide variation of milk pH as determined electronically within each BTB (Brom Thymol Blue) color score showed that the indicator results were not closely related to pH. The investigators stressed the importance of using color comparators which resemble as closely as possible the actual pH test method being utilized. If the colorimetric milk pH test results were to be interpreted immediately, it was important to use comparators that were also wetted with milk. If the calorimetric pH test results were to be determined after the milk spots had dried, it was advantageous to use dry comparators.
Returning to bacterial vaginosis, the whiff test, which is one of the Amsel criteria, originated in a study by Pheifer, et al., N. Engl. J. Med. 298:1429-1434 (1978), that reported the presence of a characteristic fishy amine odor upon the addition of 10% KOH to a vaginal fluid specimen from a woman with BV. The odor is caused by the alkaline volatilization of amine salts found in the vaginal fluid of women with BV. Unfortunately, the test is highly subjective, it exposes the health care worker to potential biological hazards, and it is disagreeable and vulnerable to error, since it is performed on a microscope slide which, due to the transient nature of the amine odor, must be placed directly under the nose and sniffed immediately after the addition of the KOH.
Alternatives to the whiff test are analytical procedures such as high voltage electrophoresis (Chen, K. C. S., et al., J. Clin. Invest. 63:828-835 (1979)), thin-layer chromatography (Chen, K. C. S., et al., J. Infect. Dis. 145:337-345 (1982), and Sanderson, B. E., et al., Br. J. Vener. Dis. 59:302-305 (1983)), gas chromatography (Gravett, M. G., et al. Obstet. Gynecol. 67:229-237 (1986), and Dravenieks, A., et al., J. Pharma. Sc. 59:495-501 (1970)), and high-performance liquid chromatography (Cook, R. L., et al., J. Clin. Microbiol. 30:870-877 (1992)). These procedures, although more accurate and reliable than the whiff test, are expensive, time-consuming, and not suitable for on-site testing in a physician""s office or clinic.
Clue cells, which constitute a further Amsel criterion, are independently correlated with BV, and in the hands of a skilled microscopist are a very sensitive and specific indication of this infection. Clue cells are squamous vaginal epithelial cells found in vaginal fluid when BV is present. The cells are covered with numerous bacteria, giving them a stippled or granular appearance, and their borders are obscured or fuzzy because of the adherence of numerous rods or cocci. According to standard clinical practice, a diagnosis of BV is established when at least 20% of the detectable epithelial cells are clue cells. Holmes, et al., Sexually Transmitted Diseases, 2d ed., McGraw-Hill, Inc., New York, 1990.
Distinguishing between true clue cells in which the adherent bacteria completely obscure the edges of the cells and cells with simply a few adherent bacteria requires training and experience. One source of error is similarity in appearance between clue cells and trichomonads, white blood cells and other vaginal fluid components, frequently resulting in an incorrect identification of these cells as clue cells, and therefore false positive test results. Another is that clue cells when present are frequently obscured by numerous vaginal fluid components causing the clinician to miss the clue cells completely or to quantify them at levels below their actual level. This can result in a false negative test result. Therefore, it would be highly desirable to have a distinct analyte that is accurately and conveniently monitored and whose presence is correlated with clue cells.
U.S. Pat. No. 5,217,444 to Schoenfeld teaches an absorbent pad, for use in absorbing secretions from a person""s body, which includes a pH indicator material indicating by a color change the acidity or alkalinity of a liquid coming into contact with it. The pH indicator material is wetted by the secretions absorbed by the pad, and thereby provides an indication of the health condition of the person""s body.
U.S. Pat. No. 5,853,669 to Wolfbeis teaches a hydrophilic accommodating layer disposed on a hydrophobic mechanically stable support element, which layer contains an indicator dye proper in an immobilized form for the purpose of visual or optical indication of the pH of a sample which can be used as the pH indicator in the pad of Schoenfeld and in other applications as well.
One object of the present invention is to improve a pH sensor of the type mentioned in the preamble and a process for fabricating a pH sensor of this type, so as to enable a simple fabrication or even mass production of pH sensors which are inexpensive and thus suitable as an expendable pH sensor.
Another object of the present invention is to provide hygienic pads in which a pH sensor as herein described is incorporated for monitoring a user""s health condition.
Still another object of the present invention is to provide an efficient method of selecting pH indicator dyes and/or dyes ratio for obtaining a pH indicator dye mixture which is sensitive to a subtle change in pH.
Thus, according to one aspect of the present invention there is provided a pH sensor for the visual or optical indication of the pH of a sample, the pH sensor comprising a hydrophilic, intrinsically charged or neutral, synthetic membrane and at least one pH indicator dye immobilized thereto, so as to prevent appreciated bleeding of the at least one indicator dye from the synthetic membrane upon immersion in an aqueous liquid.
According to another aspect of the present invention there is provided a method of manufacturing a non-bleeding pH sensor, the method comprising the step of immobilizing at least one pH indicator dye onto a hydrophilic, optionally intrinsically charged or neutral, synthetic membrane, so as to prevent appreciated bleeding of the at least one indicator dye from the synthetic membrane upon immersion in an aqueous liquid.
According to yet another aspect of the present invention there is provided an absorbent body for absorbing vaginal discharge of a person, comprising an absorbent material and a pH sensor being combined therewith for the visual or optical indication of the pH of the vaginal discharge, the pH sensor including a hydrophilic, optionally intrinsically charged or neutral, synthetic membrane and at least one indicator dye immobilized thereto, so as to prevent appreciated bleeding of the at least one indicator dye from the synthetic membrane upon immersion in an aqueous liquid, the pH sensor being included in the absorbent body so as to be wetted by the secretions absorbed by the absorbent material, thereby providing an indication of the health condition of the person""s body.
According to still another aspect of the present invention there is provided a method of providing an indication of the health condition of a person""s body, the method comprising the steps of (a) collecting vaginal discharge of the person into an absorbent body including an absorbent material and a pH sensor being combined therewith for the visual or optical indication of the pH of the vaginal discharge, the pH sensor including a hydrophilic, optionally intrinsically charged or neutral, synthetic membrane and at least one indicator dye immobilized thereto, so as to prevent appreciated bleeding of the at least one indicator dye from the synthetic membrane upon immersion in an aqueous liquid, the pH sensor being included in the absorbent body so as to be wetted by the secretions absorbed by the absorbent material; and (b) optically inspecting the pH sensor for a color change, thereby providing an indication of the health condition of the person""s body.
According to a further aspect of the present invention there is provided a method of producing an absorbent body for absorbing vaginal discharge of a person and for providing an indication of the health condition of the person""s body, the method comprising the step of combining an absorbent material and a pH sensor, the pH sensor being for the visual or optical indication of the pH of the vaginal discharge, the pH sensor including a hydrophilic, optionally intrinsically charged or neutral, synthetic membrane and at least one indicator dye immobilized thereto, so as to prevent appreciated bleeding of the at least one indicator dye from the synthetic membrane upon immersion in an aqueous liquid, the pH sensor being included in the absorbent body so as to be wetted by the secretions absorbed by the absorbent material, thereby providing an indication of the health condition of the person""s body.
According to further features in preferred embodiments of the invention described below, the at least one pH indicator dye is sensitive to a subtle change in pH of less than N pH units, wherein N is selected from the group consisting of 0.5 pH units, 0.4 pH units, 0.3 pH units, 0.2 pH units and 0.1 pH units.
According to still further features in the described preferred embodiments the hydrophilic synthetic membrane is positively charged.
According to still further features in the described preferred embodiments the hydrophilic synthetic membrane is negatively charged.
According to still further features in the described preferred embodiments the hydrophilic synthetic membrane is amphoteric.
According to still further features in the described preferred embodiments the hydrophilic synthetic membrane is a Nylon membrane.
According to still further features in the described preferred embodiments the Nylon is Nylon 6.6.
According to still further features in the described preferred embodiments hydrophilic synthetic membrane is a polyamide membrane.
According to still, further features in the described preferred embodiments hydrophilic synthetic membrane is an arylamide membrane.
According to still further features in the described preferred embodiments the at least one pH indicator dye produces a color change at a pH of between pH value of 4.0 and 5.0.
According to an additional aspect of the present invention there is provided a method of selecting pH indicator dyes and dyes ratio for obtaining a pH indicator dye mixture which is sensitive to a subtle change in pH, the method comprising the steps of (a) providing a plurality of transparent containers; (b) dividing the plurality of transparent containers into pH groups by filling containers of each of the pH groups with a buffered solution of a predetermined pH value; (c) introducing into each of the plurality of containers a single pH indicator dye of a known concentration, such that different containers of a single group of containers include different pH indicator dyes and optionally different concentrations of a specific pH indicator dye; (d) selecting at least two containers of a specific pH group, each of the at least two containers including a different pH indicator dye and positioning the at least two containers against a light source so as to optically monitor a color obtained by combining color contributions of each of the at least two containers; and (d) optionally repeating step (d) with containers of one or more of the pH groups until identifying pH indicator dyes and/or dyes ratio, such that when mixed to form the pH indicator dye mixture will be sensitive to the subtle change in pH.
According to further features in preferred embodiments of the invention described below, the subtle change in pH is of less than N pH units, wherein N is selected from the group consisting of 0.5 pH units, 0.4 pH units, 0.3 pH units, 0.2 pH units and 0.1 pH units.
The present invention successfully addresses the shortcomings of the presently known configurations by providing a pH sensor for the visual or optical indication of the pH of a sample, an method of manufacturing same, an absorbent body for absorbing vaginal discharge of a person including same, a method of providing an indication of the health condition of a person""s body using same, a method of producing an absorbent body for absorbing vaginal discharge of a person and for providing an indication of the health condition of the person""s body and a method of selecting pH indicator dyes and dyes ratio for obtaining a pH indicator dye mixture which is sensitive to a subtle change in pH.